Glioblastoma, the most malignant primary brain tumor, has remained largely refractory to all forms of therapy, and is almost uniformly lethal with a median survival of approximately 15 months. The promise of immunotherapy in glioblastoma has yet to be realized, in part due to altered immune activities in the brain and the immunosuppressive microenvironment of glioblastoma. Recently isolated glioblastoma stem cells (GSCs) are thought to be important in the tumor's ability to evade therapy. In addition, the brain tumors they generate in mice retain many of the features of the patient tumors, so they provide a representative tumor model for testing new therapies, something that established cell lines do not. We, and others have developed oncolytic herpes simplex virus (oHSV) vectors that selectively replicate in and kill cancer cells, without harming the surrounding normal tissue or causing disease, and induce anti- tumor immune responses. The safety of oHSV has been demonstrated in clinical trials for glioblastoma; however, while clinical responses have been very promising, approval for use in the brain has not yet occurred, and improvements to this therapeutic strategy are warranted. The overall goal of our research program is to develop oHSV as therapeutic agents and elucidate combination strategies with them that will cure glioblastoma. Building on the progress in the last funding cycle, we propose to improve oHSV therapy by: (i) Creating new oHSV vectors that replicate efficiently in GSCs, are safe in the brain, and are optimally immunotherapeutic; (ii) Developing new oHSV combination strategies with inhibitors of DNA damage responses to target GSCs and take advantage of oHSV disruption of DNA repair; and (iii) Enhancing glioblastoma immunotherapy by combining DNA damage response inhibitors with oHSV and immune checkpoint inhibitors. Central to these studies is the use of representative tumor models; primary and recurrent patient-derived GSCs in immunodeficient mice and mouse syngeneic GSCs in immunocompetent mice. These studies should identify new synergistic drug / oHSV combinations that also promote immunovirotherapy. We anticipate that therapies demonstrated in the proposed studies will be translatable to the clinic for glioblastoma, and other brain tumors and solid tumors in the periphery.